While the roles of cysteine as an antioxidant and in cell signaling are widely appreciated, only recently has it been recognized that methionine, like cysteine, functions as an antioxidant and as a key component of a system for regulation of cellular metabolism. The efficiency of methionine as an antioxidant or as a component of signaling systems depends on its ready interconversion between the reduced form (methionine) and the oxidized form (methionine sulfoxide). Methionine sulfoxide reductase catalyzes the reduction of methionine sulfoxide back to methionine. We reported last year that STARD3, a late endosomal/lysosomal protein binds methionine sulfoxide reductase A (MSRA) at the cytosolic fact of the late endosome. Elucidating the effect of interaction with methionine sulfoxide reductase is now a major specific aim. We have successfully created a STARD3 knockout mouse and will soon begin characterizing it. We have the MSRA knockout mouse which is being crossed onto the desired C57Bl6/N strain. This and the double knockout will also be characterized. We have also found that 4 other STARD proteins that bind cholesterol interact with MSRA. We are now investigating the interaction of STARD1, a mitochondrial protein that is essential for cholesterol transport into the mitochondria and, thus, for steroid biogenesis. It binds both myristolyated and non-myristoylated MSRA, although myristolyated MSRA is bound more tightly. We hypothesize that STARD3 and STARD1 cooperate to transfer cholesterol from the endoplasmic reticuluum to the mitochondria via an interaction with the late endosome. We want to elucidate the role of MSRA in this process. A major limitation in the field is the lack of a specific, sensitive, convenient assay of methionine sulfoxide in proteins. To this end, we have collaborated with chemists in the Imaging Probe Development Center to carry out a detailed study of the Pummerer reaction as a means to developing such an assay. A manuscript reporting these studies has been submitted. We have collaborated with the Anderson laboratory at Johns Hopkins to investigate the roles of reversible oxidative modification of CAMKII delta in cardiac physiology and pathology. Another major focus is to elucidate the mechanism by which alpha-synuclein forms covalent oligomers. Our on-going collaboration with the laboratory of Ad Bax continues to increase our understanding of the novel derivatives that arise from the interaction of DOPAL with lysine residues in alpha-synuclein. Our current emphasis is to develop specific assays to detect and quantitate these novel derivatives in vivo.